Zoosyst. Evol. 98 (2) 2022, 213-232 | DOI 10.3897/zse.98.84963 pg MuseuM TOR BERLIN Rediscovery, redescription and identity of Pristimantis nebulosus (Henle, 1992), and description of a new terrestrial-breeding frog from montane rainforests of central Peru (Anura, Strabomantidae) Jérn Kohler’, Ernesto Castillo-Urbina*, César Aguilar-Puntriano*, Miguel Vences*, Frank Glaw* Hessisches Landesmuseum Darmstadt, Friedensplatz 1, 64283 Darmstadt, Germany Universidad Nacional Mayor de San Marcos, Museo de Historia Natural, Departamento de Herpetologia, Av. Arenales 1256, Lima I1, Peru Zoological Institute, Technische Universitat Braunschweig, Mendelssohnstr. 4, 38106 Braunschweig, Germany Zoologische Staatssammlung Miinchen (ZSM-SNSB), Miinchhausenstr. 21, 81247 Miinchen, Germany FP WwW NM fF https://zoobank. org/95 93 9640-6B7F-41E9-825C-A53024113F 54 Corresponding author: Jorn Kohler (joern.koehler@himd.de) Academic editor: Pedro Taucce # Received 6 April 2022 # Accepted 12 June 2022 Published 24 June 2022 Abstract The taxonomic status of the strabomantid frog species Pristimantis nebulosus (Henle, 1992), originating from the southern Cordillera Azul in central Peru, is investigated based on examination of the holotype and its comparison with freshly collected topotypic mate- rial. Following current standards, we provide a redescription of the holotype. It is in a rather poor state and exhibits certain damages and preservation artifacts, conditions that have hampered an allocation of this nominal taxon to any known living population of Pristimantis in the past. Our detailed specimen-to-specimen comparison provided morphological evidence for the conspecifity of the holotype and freshly collected topotypes. Molecular phylogenetic analysis, based on the mitochondrial 16S gene fragment places P. nebulosus in the P. conspicillatus species group, being closely related to P. bipunctatus and an undescribed candidate species from the Cordillera de Carpish. From both, P. nebulosus differs by considerable divergence in the 16S gene (p-distance 4.1—6.2%). Based on the specimens available, we provide an updated diagnosis for P. nebulosus, compare it to other species in the P. conspicillatus group and describe its advertisement call. In addition, we describe and name the closely related candidate species from the Cordillera de Carpish. It is sister to P. bipunctatus and P. nebulosus, but differs from both mainly by its tuberculate dorsal skin (versus shagreen) and divergence in the 16S gene (3.3-4.1%). We briefly discuss cryptic species diversity in the P. conspicillatus and P. danae species groups and provide justification for the description of a singleton species. Key Words Amphibia, bioacoustics, Cordillera Azul, Cordillera de Carpish, molecular genetics, morphology, Pristimantis conspicillatus species group, systematics, taxonomy Introduction The genus Pristimantis in the anuran family Strabo- mantidae currently comprises 590 recognised species (AmphibiaWeb 2022). Species in the genus are distrib- uted from Central America southwards to southern Brazil and northern Argentina (Frost 2021), occupying a great variety of different biomes in lowland and montane areas. At present, 15 species groups are recognised in the genus (Padial et al. 2014; Gonzales-Duran et al. 2017; Paez and Ron 2019; Chavez et al. 2021; Zumel et al. 2021), but for a majority of species only sparse data are available and these, therefore, remained unassigned to any group (Padi- al et al. 2014). Furthermore, several studies indicated that species diversity in the genus is considerably underesti- mated by the number of names established, and demon- strated that resolving the taxonomy in this group might be a complex task because of intra-specific polymorphism Copyright Kohler, J. et al. This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. 214 Kohler, J. et al.: Rediscovery of Pristimantis nebulosus and description of a new species on the one hand and superficial crypsis on the other (e.g. Elmer and Cannatella 2008; Padial and De la Riva 2009; Padial et al. 2009; Ortega-Andrade et al. 2015; Lehr et al. 2017; Paez and Ron 2019). Nevertheless, the integrative application of molecular genetics, in combination with morphology and bioacoustics, has led to a continuous de- scription of new Pristimantis species at an unrestrained rate (e.g. Hutter and Guayasamin 2015; Paez and Ron 2019; Moravec et al. 2020; Carrion-Olmedo and Ron 2021; Ortega-Andrade et al. 2021). In contrast to the accelerating number of species rec- ognised in the genus Pristimantis, knowledge about many nominal species is poor and sometimes species are only known from the type specimens and have subsequently never been allocated to any known living population. One of those species is Pristimantis nebulosus (Henle, 1992). The original description of this taxon (as Eleutherodacty- lus nebulosus) was based on a single specimen collected in April 1979 by Rainer Schulte in the southern Cordillera Azul in central Peru, and the species was allocated to the E. fitzingeri species group (sensu Lynch 1976) according to a few morphological characters, such as smooth belly, first finger longer than second and tympanum distinct (Henle 1992). Subsequently, Lynch (1996) established a replace- ment name for this taxon (Eleutherodactylus caliginosus), as Henle’s (1992) name constituted a junior homonym of Syrrhophus nebulosus Taylor, 1943 (actually a secondary homonym, not a primary homonym, as stated by Lynch 1996; see ICZN 1999: Article 57.3), a taxon periodical- ly considered to be synonymous with Eleutherodactylus pipilans (Taylor, 1940) and now considered to represent a valid Central American species, E/eutherodactylus neb- ulosus (see Griinwald et al. 2021). The subsequent trans- fer of many species of E/eutherodactylus to other genera (Hedges et al. 2008) and the allocation of Henle’s taxon to Pristimantis again reestablished the species name nebulo- sus, aS a homonymy was no longer in place ICZN 1999: Article 59.4; Duellman and Lehr 2009). Since its description, Pristimantis nebulosus remained an enigmatic taxon that apparently had never been recol- lected and was rarely mentioned in the literature. Lynch (1996) and Lynch and Duellman (1997) placed it in the P. conspicillatus species group (as FE. caliginosus) and, subsequently, it was listed in systematic accounts with- out allocation to any species group (Heinicke et al. 2007; Hedges et al. 2008; Padial et al. 2014). Given that its original description (Henle 1992) is barely detailed and partly inadequate, covering less than two pages including a figure of the preserved holotype, and the fact that the type specimen is in a rather poor state of preservation, its identity and relationships remained obscure. Duellman and Lehr (2009), in their monograph on Peruvian terres- trial-breeding frogs, mentioned that, despite examination of the holotype, they were unable to associate the nomi- nal taxon with any known population of Pristimantis. During fieldwork in November 2019 in central Peru, we collected Pristimantis frogs in the Departamentos Huanu- co and Ucayali. Among the investigated sites were the zse.pensoft.net Cordillera de Carpish and the southern parts of the Cordil- lera Azul in central Peru (see Castillo-Urbina et al. 2021), the latter including the type locality of P. nebulosus. Some of the specimens collected at the P. nebulosus type locali- ty appeared to represent members of the P. conspicillatus species group and, superficially, were in agreement with the description of P. nebulosus. The present study aims at the clarification of the taxonomic status of these newly collected specimens and an evaluation of the identity of P. nebulosus, as well as the description of a closely related new species from the Cordillera de Carpish. Material and methods Fieldwork and voucher specimens Fieldwork was conducted in November 2019 in differ- ent areas of the Departamentos Huanuco and Ucayali in central Peru. Specimens were observed and collected during opportunistic searching at night using torches and headlamps, often guided by the sounds emitted by calling males. Geographic position was recorded using handheld GPS receivers set to WGS84 datum. Collected specimens were euthanised using an over- dose of 5% lidocaine gel applied on ventral surfaces of the individuals (McDiarmid 1994). Tissue samples (thigh muscle and tongue pieces) were cut prior to fixation and stored in 99% ethanol, while specimens were fixed using 96% ethanol and subsequently stored in 70% ethanol. Specimens were deposited at the herpetological collection of the Museo de Historia Natural, Universidad Nacional Mayor de San Marcos (MUSM), Lima, Peru. FGZC refers to Frank Glaw field numbers, KU refers to University of Kansas, Museum of Natural History, Division of Herpetol- ogy, Lawrence, Kansas, USA, and ZFMK refers to Zool- ogisches Forschungsmuseum A. Koenig, Bonn, Germany. External morphology Morphometric measurements (in millimetres) were taken by JK with a digital calliper to the nearest 0.1 mm. Mea- surements taken and used throughout the text are: SVL, snout—vent length; TL, tibia length; HW, head width (at level of angle of jaws); HL, head length (from posterior margin of lower jaw to tip of snout); IOD, interorbital distance; ED, horizontal eye diameter; E—N, eye—nostril distance (straight line distance between anterior corner of orbit and posterior margin of external nares); TD, hori- zontal tympanum diameter; HandL, hand length (from proximal border of outer metacarpal tubercle to tip of third finger); FootL, foot length (from proximal border of inner metatarsal tubercle to tip of fourth toe). Fingers and toes are numbered preaxially to postaxially from I- IV and I-—V, respectively. Lengths of fingers I and II were determined by adpressing the fingers against each other. For character state definitions, we followed Duellman Zoosyst. Evol. 98 (2) 2022, 213-232 and Lehr (2009). Description and diagnostic schemes fol- low Padial et al. (2016). Colouration in life was described based on digital photographs. Bioacoustics Vocalisations in the field were recorded using a Olym- pus LS-05 digital recorder with built-in microphones at 44.1 KHzand 16-bit resolution and saved as uncompressed wave format. Recordings were re-sampled at 22.05 kHz and 32-bit resolution and computer-analysed using the software Cool Edit Pro 2.0. We obtained frequency infor- mation through Fast Fourier Transformation (FFT; width 1024 points) at Hanning window function. Spectrograms were drawn with Blackman window function at 256 bands resolution. Sensitive filtering was applied to remove back- ground sounds, applied only to frequencies outside the prevalent bandwidths of calls. Temporal measurements are summarised as range with mean + standard deviation in parentheses. Description and terminology follow the call-centred scheme of Kohler et al. (2017). Taxon sampling Our genetic analysis aimed at identifying lineage diver- gence among focal lineages of Pristimantis. For represen- tative taxon sampling, we largely followed an approach as described by Castillo-Urbina et al. (2021). We compared sequences of three of our collected specimens of the focal species with available sequences of the 16S rRNA gene of Pristimantis. For this, we first blasted the sequences of the new samples against GenBank and downloaded sequenc- es with an identity > 89%. Next, we manually searched for sequences of species that are currently placed in the P. conspicillatus species group, plus representatives of the P. danae species group (sensu Padial et al. 2014), as some of the latter group share superficial morphological similari- ties with species in the P. conspicillatus group. In addition, we selected a small set of sequences of representatives as- signed to the P. /acrimosus and P. ridens species groups, as some members occur in the same general area as the focal lineages. For the P. conspicillatus and P. danae species groups, we further added seven new samples collected by us at different localities in central Peru. We unsuccessfully tried to obtain a DNA sequence from a small piece of thigh muscle from the holotype of E. nebulosus (ZFMK 27634) using the standard protocol described below. A sequence of Yunganastes pluvicanorus was included as outgroup. For details of used samples, see Appendix 1. Molecular analyses We sequenced a DNA fragment of the mitochondrial 16S rRNA gene (16S) from tissue samples of newly col- lected specimens. In brief, DNA was extracted using a 215 standard salt extraction protocol, Polymerase Chain Re- action (PCR) carried out with primers 16SAr-L (5’-CG- CCTGTTTATCAAAAACAT-3’) and 16SBr-H (5’-CCG- GTCTGAACTCAGATCACGT-3’) (Palumbi et al. 1991) and the PCR products then directly sequenced on automated DNA sequencers by LGC Genomics (Berlin, Germany). All new DNA sequences were submitted to GenBank (accession numbers ON710980—ON710989). MEGA7 (Kumar et al. 2016) was used to align sequenc- es to reference sequences of other Pristimantis down- loaded from GenBank using the Muscle algorithm (total resulting alignment length 584 bp), and to identify the GTR+I+G substitution model as best fitting the data set under the Bayesian Information Criterion. We used this substitution model to infer a Maximum Likelihood (ML) tree in RAXML v8 (Stamatakis 2014) as implemented in raxmlGUI v2.0 (Edler et al. 2021), assessing node sup- port with 500 thorough non-parametric ML bootstrap replicates. To quantify genetic divergences, we calculat- ed uncorrected pairwise distances among the sequences (p-distances) in MEGA7. Results and conclusions The holotype of Eleutherodactylus nebulosus Henle, 1992 We examined the holotype of Eleutherodactylus nebulo- sus (ZFMK 27634) in detail. As former authors already mentioned (Duellman and Lehr 2009), we confirm that the Specimen is in a very poor state. The body is rather soft, whereas the head and distal parts of fingers and toes are hardened and completely desiccated. We speculate that the specimen once was almost completely dry and then after immersion in preservation liquid partly softened. Such phenomena may occur if, for example, the collected indi- vidual died in the collection bag due to heat and is trans- ferred to preservation liquid a certain time after without proper fixation. Judging from the figure provided by Henle (1992), the holotype was already in this poor condition at the time of its description. This fact may have resulted in the description of preservation artifacts erroneously con- sidered to represent morphological traits by Henle (1992). To somehow quantify the condition of the type specimen, we in the following list the most obvious artifacts and dam- ages: (1) vertebral column broken close to urostyle, result- ing in body being longitudinally compressed, with virtually shorter SVL; urostyle bones protruding dorsally; (2) head desiccated, lacking almost all fleshy parts, dorsolaterally compressed and squeezed, resulting in virtually greater HW; (3) distal parts of fingers completely desiccated; and (4) distal parts of toes completely desiccated (Fig. 1). We here provide a redescription of the holotype of Eleutherodactylus nebulosus Henle, 1992 (ZFMK 27634) using current standards, considering that several charac- ter descriptions are to be treated with some caution or are not accessible (NA) due to the poor state of preservation. zse.pensoft.net 216 Kohler, J. et al.: Rediscovery of Pristimantis nebulosus and description of a new species Figure 1. Preserved holotype of Eleutherodactylus nebulosus Henle, 1992 (ZFMK 27634) in a. dorsal and b. ventral views; c. Palmar surface of left hand; d. Head in lateral profile. Note that due to the poor state of preservation and damage, certain characters appear to be altered, including body shape, finger length, finger discs, and head shape. Eleutherodactylus nebulosus Henle, 1992 Redescription of the holotype. An adult male (Fig. 1) with subgular vocal sac and vocal slits. SVL 28.6 mm (for additional measurements see Table 1). Head slightly longer than wide (HL/HW = 1.1); snout shape NA; nos- trils oriented posterolaterally; canthus rostralis straight in dorsal view, sharp in profile; loreal region very slight- ly concave; lips not flared; upper eyelid without tuber- cles; cranial crests absent. Supratympanic fold prom- inent, long, curved, surrounding the upper tympanic membrane; tympanic membrane and annulus distinct; tympanic membrane nearly round on the left side, slight- ly higher than long on right side, its length less than half the eye diameter; two prominent postrictal tubercles, conical. Choanae not concealed by palatal shelf of the maxillary arch when roof of mouth is viewed from be- low; choanae large, ovate, separated by distance equal to six times diameter of a choana; dentigerous process zse.pensoft.net of vomers prominent, triangular in shape, not in contact, oblique, situated posteromedial to choanae; vocal sac subgular, vocal slits placed posterolaterally. Skin of dor- sal surfaces shagreen, with few scattered low tubercles; skin on throat, chest and belly weakly areolate, almost smooth; occipital folds absent: dorsolateral folds pres- ent, low, formed by a discontinuous row of anastomosed granules; discoidal fold conspicuous. Arm without ulnar tubercles; palmar tubercle bifid, relatively flat, equal to elongate thenar tubercle; few supernumerary tubercles present, low, round, smaller than subarticular tubercles; subarticular tubercles prominent, subconical; finger tips enlarged (NA completely due to desiccation and shrink- ing); fingers without lateral fringes; basal webbing be- tween fingers IT and III; relative length of fingers HI > IV > II V>IUI > Il >I; toe V reaching proximal level of penultimate subarticular tubercle of toe IV. Tibiotarsal articulation reaches far beyond tip of snout when hind limb flexed parallel to axis of body; heels broadly over- lapping when hind limbs flexed perpendicular to axis of body. After 42 years in preservative (Fig. 1), the dorsal sur- faces are overall tan to brown of different shades, with dark brown chevrons outlined with cream; arms and legs are barred with brown bars the same colour as those of dorsum and the same colour applies to vertical bars on the flanks, which are also outlined with cream; dark brown interorbital bar; canthus rostralis darker than surround- ing skin; triangular cream coloured fleck on tip of snout; supratympanic fold and a pair of occipital spots black; posterior surfaces of thighs brown, with fine scattered ir- regular cream spots; chest and belly greyish-white with some scattered irregular brown flecking on chest; throat cream-coloured, but densely mottled with fine brown to grey spots and flecking which (like those on chest) are rather distinct and contrasted; lower and upper lips brown and barred with cream; soles of hands and feet brown with irregular cream flecking. Our examination of the type specimen revealed the following discrepancies to the original description: Henle (1992) described the first finger being longer than the sec- ond. Although this appears to be correct at first view (see Fig. 1), it is to be mentioned that the tip of finger II is al- most completely desiccated and thus the finger apparently lost some of its total length. However, even at this state of preservation, when fingers I and II are adpressed against each other, they are about equal in length. At most, we 217 could state that finger I is only slightly longer than finger II. Henle (1992) described the terminal finger discs as be- ing only slightly expanded. As stated above, distal fingers of the holotype are completely desiccated and apparently were already during the time of the species description (Henle 1992: fig. 2). As a consequence, finger discs are barely obvious, but when viewed with magnification, T-shaped terminal phalanges are clearly visible, indi- cating the probable former presence of expanded finger discs. The original description (Henle 1992) stated that an outer metatarsal tubercle is lacking. The desiccation event apparently resulted in less distinct and less elevat- ed tubercles on hands and feet (including subarticular tubercles) in the holotype, but with strong magnification (> 20x), skin structures become evident which likely once represented a flat outer metatarsal tubercle. Moreover, Henle (1992) described the tympanum as being slightly higher than long, a state we were able to confirm only for the right body side of the holotype, whereas it exhibits a nearly round tympanum on the left body side (Fig. 1). We reject Henle’s (1992) statement that toes exhibit webbing of about one fourth of their length, as only basal webbing is present between the toes of the holotype. The supra- tympanic fold has been described as weakly developed by Henle (1992), but it is actually rather distinct in the holotype. Due to the condition of the type specimen, we were unable to confirm or reject Henle’s (1992) charac- terisation of the snout shape. As mentioned above, fleshy parts are almost completely lacking at the anterior head of the holotype and the shape of the head in its current condition seems to be basically represented by the skull shape. Moreover, the head shape has likely been altered by its dorsoventral compression and squeezing. New topotypic specimens In the the original description of P. nebulosus, the type locality 1s given as “Departamento Huanuco, Cordillera Azul, PaB der Carretera Central auf 1650 m NN” (Henle 1992), which translates to Departamento Huanuco, Cor- dillera Azul, pass of the Carretera Central at 1650 m above sea level. Henle (1992), in his itinerary, called this same locality “Divisoria, Cordillera Azul (1650)”. During our fieldtrip, we searched the surroundings of a spot locally called ‘Abra La Divisoria’ (9.20017°S, 75.79531°W), a pass of the national road 5N at 1650 m a.s.l. at the bor- der of the Departamentos Huanuco and Ucayali, where a small restaurant and fuel station are located. This lo- cality is not to be confused with the small village called ‘Divisoria’, located few kilometres eastwards and down- hill along the same road, situated already in Departamen- to Ucayali. According to the concordant elevation, pres- ence of a road pass and the name of the locality, we have no doubts that ‘Abra La Divisoria’ actually constitutes the type locality of P. nebulosus. At Abra La Divisoria, we collected two adult males (MUSM 40298-40299, FGZC 6253-6254; coll. 8 zse.pensoft.net 218 Kohler, J. et al.: Rediscovery of Pristimantis nebulosus and description of a new species November 2019) and one juvenile (MUSM 40326, FGZC 6281; coll. 10 November 2019) of a species of Pristimantis which, due to their general appearance, could be identified as members of either the P. conspicil- latus or the P. danae species group (sensu Padial et al. 2014). These three specimens had identical sequences of the 16S gene (see below for molecular relationships) and, thus, are to be considered conspecific. Despite un- clear relationships, P. nebulosus has been associated with the P. conspicillatus species group by Lynch (1996) and Lynch and Duellman (1997), which led us to hypothesise that the newly collected specimens might constitute a re- discovery of this enigmatic species. Given that the origi- nal species description (Henle 1992) is barely helpful for proper identification, we compared our collected speci- mens with the holotype of E. nebulosus in detail. At first superficial examination, the holotype seemed to differ from our specimens by a stouter body shape and broad- er head. However, as described above, the poor state of preservation and certain damage apparently are reasons for this particular appearance. Our detailed comparison of morphological characters did not reveal any diagnos- tic differences between the holotype and our specimens which are not explainable by damage, preservation arti- facts, or intra-specific variation. Our collected adult male specimens (Figs 2, 3) gen- erally agree morphologically with the male holotype of E. nebulosus as described above in all diagnostic char- acters, namely: similar size and overall proportions (see Table 1 for measurements); skin on dorsum shagreen with few scattered small tubercles, skin of throat and venter weakly areolate; discoidal fold conspicuous; indistinct, low dorsolateral fold; tympanum and tympanic annulus distinct, its diameter slightly less than half of eye di- ameter, nearly round; supratympanic fold prominent, curved, surrounding upper tympanum; two prominent conical postrictal tubercles; occipital folds absent; head slightly longer than wide; canthus rostralis straight in dorsal view, sharp in profile; dentigerous process of vom- ers prominent, triangular in outline, oblique, situated Figure 2. Preserved topotypic male of Pristimantis nebulosus (MUSM 40299, FGZC 6254) from Abra La Divisoria, Cordillera Azul, 1650 maz.s.l., Peru, in a. dorsal, and b. ventral views; ce. Palmar surface of left hand; d. Head in lateral profile. zse.pensoft.net Zoosyst. Evol. 98 (2) 2022, 213-232 Figure 3. Topotypic Pristimantis nebulosus in life: Adult male (MUSM 40299) in a. dorsolateral, b. ventral, c. dorsal, and d. frontolateral views; adult male (MUSM 40298) in e. dorsolateral, f. ventral, and g. frontal views; h. Juvenile (MUSM 40326) in dorsolateral view (inset photo showing ventral view of same individual). Not to scale. zse.pensoft.net 220 Kohler, J. et al.: Rediscovery of Pristimantis nebulosus and description of a new species posteromedial to choanae; first finger only very slightly longer than second, almost equal in length; lateral fringes on fingers absent, basal webbing between fingers II and II; subarticular tubercles prominent, conical; palmar tu- bercle bifid, thenar tubercle ovoid; terminal finger discs expanded; toes long and slender, basal webbing between toes; toe V slightly longer than toe HI; inner tarsal tu- bercle elongated, prominent; outer metatarsal tubercle inconspicuous, ovoid; terminal toe discs expanded; dor- sum with dark chevron-shaped markings, outlined with cream; a pair of occipital black spots; dark interorbital bar; flanks with dark diagonal bands and upper surfaces of arms and thighs with dark crossbands, all outlined with cream; triangular cream-coloured fleck on tip of snout; throat with dark spotting and mottling, chest and belly cream with some scattered dark flecks anteriorly; ventral surfaces of thighs with dark spotting; posterior surfaces of thighs dark with fine cream spotting; upper and lower lip dark with irregular cream spots and bars; broad black supratympanic stripe. Despite this congruence in character states, some ob- servations are in need of further comments. The shape of terminal discs on fingers is almost undiscoverable in the holotype due to complete desiccation and shrinking. However, given the presence of clearly T-shaped terminal phalanges in the type specimen, we are convinced that it actually exhibited distinctly expanded finger tips as present in our fresh material. The ground colouration of the preserved holotype is considerably darker and more brownish compared to our adult males (predominantly greyish with some tan brown to reddish tint in preser- vative). This is particularly obvious with respect to the less contrasting pattern of dark spots and mottling on throat and chest (compare Figs 1, 2). This ventral pat- tern is brown, rather contrasting and conspicuous in the holotype, while it is grey and appears less dense and less contrasting in our specimens. Nevertheless, the general pattern of ventral markings is the same among all speci- mens, with the throat most densely mottled, mottling and flecking present on chest and more widely spaced fleck- ing on the anterior belly. Overall, the holotype is distinct- ly darker coloured and brownish when compared to our preserved greyish specimens, but the more contrasting pattern on the dorsum and venter may most likely be ex- plained as a result of the preservation method used for the holotype (which remains unknown). Although only few specimens were available, variation is particularly evident in the colour pattern. The three specimens col- lected by us differ in the extent and shape of dark dorsal markings. Remarkably, the juvenile specimen (MUSM 40326) is considerably darker in ground colour compared to the two adult males and has a more contrasting pat- tern (Fig. 3), thus very strongly resembling the colour and pattern of the holotype. Another difference constitutes the presence of a broad dark canthal stripe in our specimens, not evident in the holotype. Given that the canthal region in the holotype is slightly darker coloured compared to the surrounding skin, particularly on the right side, we zse.pensoft.net suspect that this pattern simply faded. This assumption might appear unjustified, as otherwise the colour pattern of the holotype is more contrasting compared to the fresh- ly collected material, but obviously the head of the holo- type body suffered most from desiccation which might have also promoted colour fading to a greater extent. In summary, our morphological comparison revealed overall congruence in character states between the holo- type and newly collected topotypes. Differences observed can either be explained by damage and preservation arti- facts, or intra-specific variation, respectively. We finally conclude that the specimens collected by us at the type locality of P. nebulosus are conspecific with the respec- tive holotype. Identity of Pristimantis nebulosus Pristimantis nebulosus (Henle, 1992) Relationships. The Maximum Likelihood tree, based on an alignment of 584 bp of the 16S gene, is insufficient to establish a reliable hypothesis of phylogenetic relation- ships among the Pristimantis samples included (Fig. 4). The P. conspicillatus species group is recovered para- phyletic with respect to the sample of P gaigeae from Panama, a member of the P. conspicillatus group sensu Hedges et al. (2008) and Padial et al. (2014). Together with species representing the P. lacrimosus, P. ridens and P. danae species groups, P. gaigeae is placed sister to the remaining species of the P. conspicillatus group. Within the P. conspicillatus group, most relationships are not resolved and basal nodes did not receive signif- icant support. However, the three specimens of the focal P. nebulosus are recovered with high support (bootstrap value 98%) as members of a clade containing nominal P. bipunctatus and an undescribed candidate species, with P. nebulosus placed sister to nominal P. bipunctatus (without significant support; 36%). The clade containing P. nebulosus, P. bipunctatus, and the candidate species are recovered sister (76%) to a clade containing two sam- ples of P. skydmainos (a close relationship of P. bipunc- tatus and P. skydmainos has already been revealed by Padial et al. 2014). Our P. nebulosus specimens had un- corrected p-distances in the studied 16S gene fragment of 5.4-6.2% to P. bipunctatus (with the greatest distance of 6.2% referring to the P. bipunctatus holotype KU291638) and 4.1% to the mentioned candidate species (Table 2). P-distances to other species in the P. conspicillatus spe- cles group range from 5.8—17.8%. Diagnosis. Based on the holotype and the newly collected material, we provide the following updated diagnosis for Pristimantis nebulosus (Henle, 1992): a medium-sized species of the Pristimantis conspicillatus Species group (based on molecular relationships and mor- phological similarity), with 27.7—-29.1 mm SVL in adult males (n = 3), characterised by: (1) skin on dorsum sha- green, with few scattered small tubercles; flanks shagreen; Zoosyst. Evol. 98 (2) 2022, 213-232 221 100| Pristimantis bipunctatus KYO06090 Peru: Pui Pui Forest Pristimantis bipunctatus KY594758 Peru: Pui Pui Forest Pristimantis bipunctatus EF493702 Peru: E of Oxapampa 99" Pristimantis bipunctatus KY006089 Peru: PN Yanachaga-Chemillén Pristimantis nebulosus FGZC 6254 Peru: Abra La Divisoria —_____ gg 100] Pristimantis nebulosus FGZC 6253 Peru: Abra La Divisoria Pristimantis nebulosus FGZC 6281 Peru: Abra La Divisoria Pristimantis symptosus sp. nov. FGZC 6207 Peru: Cordillera de Carpish —__ Pristimantis cf. skydmainos ZSM 1980/2008 Peru: Panguana Pristimantis skydmainos EF493393 Peru Pristimantis gutturalis JN691313 French Guiana: Mitaraka << Pe Pristimantis incertus EU186650 Trinidad and Tobago: King's Bay Recent Pristimantis zeuctotylus JN691259 French Guiana: Angouleme ef Pristimantis vilarsi KP149278 Colombia: Finca Los Almendros 6s-_ Pristimantis fenestratus KU495458 Brazil: Manaus 26| — Pristimantis fenestratus KY712673 Brazil: Borba Pristimantis koehleri EU192278 Bolivia: Angostura-Samaipata road Pristimantis samaipatae EU192292 Bolivia: Angostura-Samaipata road Pristimantis latro KX242519 Brazil: Anapu Be Pristimantis chiastonotus EU201061 Suriname: Brownsberg Pristimantis vinhai MN954211 Brazil: PN Serra das Lontas 98 Pristimantis palodutrai JX267360 Brazil: Canavieiras a Pristimantis ramagii JX267319 Brazil: Murici 49 Pristimantis lymani EF493392 Ecuador: 3.9 km E of Loja Pristimantis achatinus MK829664 Ecuador: Valle Hermoso 100] Pristimantis malkini MH516185 Ecuador: Jatun Sacha 99 98 76 100 81 96 , Pristimantis malkini EU186663 Ecuador: Monte Tour 88 Pristimantis citriogaster EF493700 Peru: 14 km NE of Tarapoto 49 Pristimantis condor EF493701 Ecuador: N of Galaquiza 56 Pristimantis conspicillatus EF493529 Ecuador: Sucumbios 87 Pristimantis buccinator EU712630 Peru: Blanquillo 100" Pristimantis buccinator KY652650 Peru: Los Amigos Biological Station Pristimantis rupicola MN954206 Brazil: Serra do Buriti do Ouro Pristimantis gaigeae FJ784461 Panama: PN Omar Torrijos Pristimantis pulchridormientes KX664107 Peru: PN Tingo Maria es Pristimantis pluvialis KX155577 Peru: Kosnipata near Chontacha Pristimantis lacrimosus MT636524 Ecuador: Pastaza Pristimantis bromeliaceus MT636527 Ecuador: Morona Santiago Pristimantis lanthanites EF493695 Peru: San Jacinto a Pristimantis colomai EF493354 Ecuador: Alto Tambo Pristimantis toftae EF493353 Peru: 15 km E of Puerto Maldonado Pristimantis rhabdolaemus EU192257 Bolivia: La Yunga de Mairana dnoi6 saioads snjeyjinidsuoo Y 82 68 a Pristimantis danae EU192270 Peru: Kosnipata valley i Pristimantis danae EU192265 Bolivia: Serrania Bella Vista 62 Pristimantis reichlei KY652657 Peru: Kosnipata valley °°" Pristimantis reichlei EU192288 Peru: Qince Mil Pristimantis reichlei EU192287 Bolivia: Los Guacharos °/'— Pristimantis reichlei EU192285 Peru: entre Puerto Leguia y San Gaban Pristimantis cf. reichlei ZSM 1985/2008 Peru: Panguana A Pristimantis cf. reichlei ZSM 1986/2008 Peru: Panguana ® 5 -4 Too| Pristimantis cf. reichlei FGZC 5342 Peru: Panguana 22 pee Pristimantis cf. reichlei F6ZC 6334 Peru: Panguana Qa Pristimantis cf. reichlei FGZC 3300 Peru: Panguana S Figure 4. Maximum Likelihood tree inferred from a 584 bp fragment of the mitochondrial 16S gene of Pristimantis species. Values at nodes are bootstrap proportions in percent (values below 49% not shown). Newly obtained sequences for this study are in red font. For other samples used, species names are followed by GenBank accession number and locality. Yunganastes pluvicanorus was used as outgroup (not shown for graphic reasons only). Inset photos depict Pristimantis bipunctatus (MUSM 31120), P. nebulosus (MUSM 40299, FGZC 6254) and P. symptosus sp. nov. (MUSM 40256, FGZC 6207) in life. zse.pensoft.net 222 Kohler, J. et al.: Rediscovery of Pristimantis nebulosus and description of a new species Table 2. Estimates of uncorrected p-distances in a 491 bp fragment of the mitochondrial 16S gene among selected samples of the Pristimantis conspicillatus species group, calculated with MEGA7 (all ambiguous positions were removed for each sequence pair). FGZC 6253 P. nebulosus FGZC 6253 (Abra La Divisoria) P. nebulosus FGZC 6254 (Abra La Divisoria) 0.000 P. nebulosus FGZC 6281 (Abra La Divisoria) 0.000 P. symptosus FGZC 6207 (Cordillera de Carpish) 0.041 P. bipunctatus KU 291638 (E of Oxapampa) 0.062 P. bipunctatus MUSM 31179 (Pui Pui Forest) 0.054 P. bipunctatus MUSM 31120 (Yanachaga Chemillén) 0.058 P. bipunctatus NMP6V/75062 (Pui Pui Forest) 0.054 throat, chest and venter weakly areolate, smooth only in the middle and anterior part; posterior surfaces of limbs smooth; discoidal fold conspicuous; dorsolateral folds present, but low, partly interrupted; dorsal folds absent; two prominent postrictal conical tubercles present; up- per eyelid lacking tubercles and granules; (2) tympanic membrane and annulus distinct, nearly round, their length slightly less than half of eye diameter; supratympanic fold long, prominent, curved, surrounding upper tympanum; (3) head slightly longer than wide; snout subacuminate in dorsal view, rounded in lateral profile; canthus rostralis straight in dorsal view, sharp in profile; (4) cranial crests absent; (5) dentigerous process of vomers large, triangu- lar in outline, oblique, situated posteromedial to choanae; (6) males with vocal slits, single subgular vocal sac, and nuptial asperities on dorsal surface of thenar tubercle; (7) hands with long and slender fingers, first finger only very slightly longer than second; subarticular tubercles subconical, prominent; supernumerary tubercles round, smaller than subarticular tubercles; palmar tubercle bi- fid; thenar tubercle elongated; terminal discs of inner two fingers enlarged and round, those of external fingers en- larged, ovate to triangular, about twice the width of digit proximal to disc; circumferential grooves conspicuous, ungual flap slightly indented; lateral fringes and keels on fingers absent; basal webbing between fingers II and III; (8) ulnar tubercles absent; (9) tubercles on heel and tarsus absent, tarsal fold present, narrow and oblique; (10) inner metatarsal tubercle ovate, prominent; outer metatarsal tu- bercle round, flat, inconspicuous; supernumerary tuber- cles absent; (11) toes long and slender (FootL 54-56% SVL); narrow lateral fringes weakly expressed, basal toe webbing present; toe V reaching mid-level of penulti- mate subarticular tubercle of toe IV; toe V slightly longer than toe III; tips of toes rounded to slightly truncate, en- larged; circumferential grooves evident, ungual flap not indented; (12) in life, dorsal colouration overall brown of different shades (Sometimes with orange to reddish tint), with dark brown chevrons or irregular flecks on dorsum, outlined with cream; dark brown bars on dorsal surfaces of arms and legs, outlined with cream; brown interorbit- al bar, at least anteriorly outlined with cream; a pair of black occipital spots: broad black supratympanic stripe; greyish-black canthal stripe; lips dark brown, barred with cream; tip of snout with triangular cream-coloured zse.pensoft.net FGZC FGZC FGZC KU MUSM MUSM 6254 6281 6207. 291638 31179 31120 0.000 0.041 0.041 0.062 0.062 0.041 0.054 0.054 0.033 0.037 0.058 0.058 0.039 0.006 0.031 0.054 0.054 0.033 0.037 0.000 0.031 fleck; belly yellowish-cream; throat yellow or yellow- ish-cream, mottled with grey spots; ventral surfaces of thighs and shanks orange brown to reddish-brown; pos- terior surface of thighs reddish-brown with small light spots; iris bronze, with some dark brown reticulation and a median reddish-brown streak; posterior iris periphery turquoise; bones white; (13) advertisement call consist- ing of a single pulsed note of 206-382 ms duration with a pulse rate of 156—174 pulses/second, emitted in short series (see below). Comparison. Among the now 41 recognised species in the P. conspicillatus species group (Padial et al. 2014, 2016; de Oliveira et al. 2017, 2020; Taucce et al. 2020), 12 species, including P nebulosus, exhibit dorsolater- al folds (Duellman and Lehr 2009; Padial et al. 2016). Among these, P. avicuporum mainly differs by the pres- ence of an interocular dermal fold, fingers bearing later- al fringes, and an upper eyelid with numerous tubercles (Duellman and Pramuk 1999; Duellman and Lehr 2009); P. buccinator mainly differs by continuous and prominent dorsolateral folds, an X-shaped or V-shaped dorsal fold and presence of an interorbital dermal ridge (Rodriguez 1994): P. condor mainly differs by larger male size (SVL 32.1-39.5 mm), finger I distinctly longer than finger II, lack of basal webbing between fingers II and III, and lack of basal webbing between toes (Lynch and Duellman 1980); P. conspicillatus mainly differs by finger I being distinctly longer than finger II, fingers with lateral keels, lack of basal webbing between fingers II and III, and pos- terior surfaces of thighs with orange spots in life (Duell- man and Lehr 2009); P. malkini mainly differs by slightly larger male size (SVL 30.4—37.2 mm), finger I distinct- ly longer than finger II, and more extensive webbing between toes (Lynch 1980; Duellman and Lehr 2009); P. meridionalis mainly differs by finger I being shorter than finger II, dentigerous processes of vomers small and round, and fingers bearing lateral fringes (Duellman and Lehr 2009); P. peruvianus mainly differs by prominent and continuous dorsolateral folds, finger I distinctly lon- ger than finger IJ, fingers with lateral fringes, and lack of basal webbing between toes (Kohler 2000; Padial and De la Riva 2009); P. skydmainos mainly differs by more prominent and continuous dorsolateral folds, finger I be- ing shorter than finger II, and a black mid-dorsal tubercle (Flores and Rodriguez 1997; Padial and De la Riva 2005). Zoosyst. Evol. 98 (2) 2022, 213-232 Superficially, P. nebulosus is similar to P. iiap, with which it shares similar adult male size, inconspicuous and partly interrupted dorsolateral folds, finger I only very slightly longer or equal in length to finger II, presence of postric- tal tubercles, and similar colour pattern (Padial et al. 2016). However, P. iiap mainly differs by the lack of bas- al webbing between fingers II and III (present in P. nebu- losus), lack of basal toe webbing (present), upper eyelid with small granules (absent), a copper-coloured iris in life (bronze), and advertisement call (Padial et al. 2016; see below). The closely related species P. bipunctatus also shares many characters with P. nebulosus, such as similar male size, dorsolateral folds present but inconspicuous, finger I about equal in length to finger II, upper eyelid lacking tubercles or granules, and similar colour pattern. However, it differs from P. nebulosus by the lack of bas- al webbing between fingers II and III (present), bluntly rounded snout in dorsal view (subacuminate), truncate in profile (rounded), basal webbing between toes IV and V (basal webbing between all toes) (Duellman and Hedges 2005; Duellman and Lehr 2009), and considerable diver- gence in the mitochondrial 16S gene. According to Du- ellman and Lehr (2009), P. adiastolus is morphologically cryptic to P. bipunctatus. However, P. adiastolus mainly differs from P. nebulosus by more prominent and continu- ous dorsolateral folds, lack of basal webbing between fin- gers, and finger I slightly shorter than finger IT (Duellman and Hedges 2007). Individuals of the recently described species P. pictus, P. pluvian and P. moa from Amazonian Brazil may sometimes exhibit weakly developed dorso- lateral folds, but all differ from P nebulosus by smooth mid-venter (versus areolate) and advertisement call (de Oliveira et al. 2020). Natural history. Pristimantis nebulosus occurs in an area of evergreen montane rainforest of moderate height. Forest grows at moderate to steeply sloped terrain (Fig. 5). See also Myers and Daly (1979) for a detailed description of the area. The area has been heavily altered by differ- ent kinds of plantations (e.g. tea). Individuals were found in disturbed habitat along a narrow dirt road. Males were Figure 5. View of the forest at the type locality of Pristimatis nebulosus at Abra La Divisoria, southern Cordillera Azul, De- partamento Huanuco, Peru, at 1650 m a.s.l. Photo taken on 8 November 2019. 223 calling from a bushy area during light rain at night, one in- dividual called from near the ground, another one was sit- ting on a leaf about 30 cm above the ground. The species was found in syntopy with Pristimantis sp. (aff. divnae), P. sp. (lacrimosus group), Rhinella sp. (margaritifera group), Boana lanciformis, Dendropsophus aperomeus, Scinax garbei, S. sp. (aff. ruber) and Adenomera sp. Vocalisation. Calls were recorded on 8 November 2019, around 20:00 h, at Abra la Divisoria, Departamen- to Huanuco, 1650 m a.s.l. (air temperature estimated 18 °C). The recorded individual could not be observed call- ing, but searching at the spot of sound emission revealed an individual of P nebulosus, leaving little doubt that recorded calls actually correspond to this species. The advertisement call consists of a single regularly pulsed note, repeated at a rather regular succession in short call series (Fig. 6). Call series consist of seven calls (n = 2), with the initial call being the longest in duration and sep- arated from the remaining calls of the series by a longer inter-call interval with about two times the duration of other inter-call intervals. Pulses within calls (= notes) are broadly fused, but fairly countable in the oscillogram. Apart from the pulse structure, there 1s amplitude mod- ulation within each call, with the greatest amplitude oc- curring at the beginning of the call, then decreasing to about half the initial amplitude at about one third of the call’s duration and rapidly decreasing to zero at the call’s end, giving the call a bell-shaped appearance in the oscil- logram. Numerical parameters of 14 analysed calls (two call series) of one male are as follows: call duration (= note duration) 206-382 ms (254.1 + 40.3 ms); inter-call Frequency (kHz) Relative amplitude 250 500 750 1000 Time (ms) Relative amplitude 0 2000 4000 6000 8000 Time (ms) Figure 6. On top, audiospectrogram and corresponding oscillo- gram of one advertisement call of a topotypic Pristimantis neb- ulosus from Abra la Divisoria, Cordillera Azul, 1650 m, Peru, at 1000 ms time scale. Below, an oscillogram at 8000 ms time scale showing one complete call series consisting of 7 calls of the same individual. Recording band-pass filtered at 450-9600 Hz. zse.pensoft.net 224 Kohler, J. et al.: Rediscovery of Pristimantis nebulosus and description of a new species interval within call series 507-1730 ms (796.1 + 348.8 ms); pulses per call 33-37 (35.4 + 1.5); pulses per second 156-174 (162.8 + 7.0); dominant frequency 3186-3359 Hz (3230 + 57 Hz); prevalent bandwidth 1500-6600 Hz, but bands of very low call energy recognisable up to 9600 Hz. Apart from the dominant frequency band, addition- al frequency bands, apparently induced by the pulse rate (not harmonics; see Kohler et al. 2017), occur at approx- imately 1400-1900, 4400-5000, 6000-6600, and 7900— 8400 Hz, respectively. Within regular call series, calls were repeated at a rate of 54—72 calls/minute. Duration of call series was 7.13 and 6.05 seconds, respectively. Distribution. So far, P. nebulosus is only known from its type locality in the southern Cordillera Azul, at the border of the Departamentos Huanuco and Ucayali. How- ever, it is very likely that the species occupies a wider range, at least along the same elevational corridor within the Cordillera Azul. Possibly, there are additional records of this species represented by unidentified or misidenti- fied specimens in scientific collections. Description of a new species During our field trip in November 2019, we collected an individual of Pristimantis at the eastern slope of the Cor- dillera de Carpish in Departamento Huanuco, which we tentatively identified as P. cf. bipunctatus. We included a 16S sequence of this specimen in our molecular analysis. It turned out to represent a member of the P. conspicil- latus species group, being sister to a clade containing nominal P. bipunctatus and P. nebulosus, although rela- tionships among the samples of this subclade are not fully resolved (Fig. 4). However, this specimen exhibits con- siderable differentiation in the 16S gene when compared to P. bipunctatus (p-distances ranging from 3.3-4.1%, with the highest value of 4.1% referring to the holotype of P. bipunctatus KU 291638) and P. nebulosus (p-distance 4.1%; see Table 2). Detailed morphological examination revealed qualitative differences in character states when compared to known species in the P. conspicillatus group and provided further evidence for its distinctiveness. Therefore, we describe this specimen as a new species. Pristimantis symptosus sp. nov. https://zoobank. org/26DBAFEE-66A D-4416-95CC-4A A DBD6CD23E Holotype. MUSM 40256 (FGZC 6207), adult male, from a point along the road 18A, approximately 5.5 km air- line distance northeast of the Carpish Tunnel (9.67744°S, 76.07313°W, 2360 m a.s.l.), Cordillera de Carpish, De- partamento Huanuco, Peru, collected on 4 November 2019 by E. Castillo-Urbina, F. Glaw, and J. Kohler. Diagnosis. A medium-sized species of the Pristiman- tis conspicillatus species group (based on molecular re- lationships and morphological similarity), with 27.6 mm SVL in adult male, characterised by: (1) skin on dorsum zse.pensoft.net tuberculate, with a pair of enlarged scapular warts; flanks tuberculate; throat smooth, venter weakly areolate; dis- coidal fold conspicuous; dorsolateral folds distinct, but low; dorsal folds absent; three prominent postrictal con- ical tubercles present; upper eyelid lacking tubercles and granules; posterior surfaces of thighs smooth; (2) tym- panic membrane and annulus distinct, slightly higher than long, their length less than half of eye diameter; supra- tympanic fold long, prominent, almost straight, not cov- ering upper tympanum or annulus; (3) head longer than wide; snout subacuminate in dorsal view, bluntly rounded in lateral profile; canthus rostralis straight in dorsal view, sharp in profile; (4) cranial crests absent; (5) dentigerous process of vomers prominent, elongate, oblique, situat- ed posteromedial to choanae; (6) males with vocal slits, single subgular vocal sac, and nuptial asperities on dor- sal surface of thenar tubercle; (7) hands with long and slender fingers, first finger equal in length to second; subarticular tubercles subconical, prominent; supernu- merary tubercles absent; palmar tubercle bifid, flat; the- nar tubercle prominent, elongated; terminal discs of inner two fingers enlarged and round, those of external fingers enlarged, ovate, about twice the width of digit proximal to disc; circumferential grooves conspicuous, ungual flap very slightly indented; lateral fringes on fingers absent; basal webbing between fingers absent; (8) ulnar tubercles absent; (9) tubercles on heel and tarsus absent, tarsal fold absent; (10) inner metatarsal tubercle ovate, prominent; outer metatarsal tubercle round, flat; supernumerary tu- bercles absent; (11) toes long and slender (FootL 54% SVL); narrow lateral fringes weakly expressed, trace of basal toe webbing present; toe V reaching distal level of penultimate subarticular tubercle of toe IV; toe V slightly longer than toe III; tips of toes rounded to slightly ovate, enlarged; circumferential grooves conspicuous; (12) in life, dorsal colouration brown to tan with dark brown chevrons and flecks on dorsum; dark brown bars on dor- sal surfaces of arms and legs; dark brown interorbital bar; a pair of black spots, surrounding prominent conical scapular warts: broad black supratympanic stripe; black canthal stripe; belly cream; throat densely mottled with dark brown; ventral surfaces of thighs and shanks orange tan; posterior surface of thighs orange-brown with irreg- ular cream spotting; plantar and palmar surfaces yellow- ish-brown, densely covered with dark brown mottling; iris copper, with black reticulation; posterior iris periph- ery pale blue; bones white; (13) advertisement call con- sisting of a single pulsed note of 132—186 ms duration, emitted at regular succession (see below). Comparison. Among the now 42 recognised spe- cies in the P. conspicillatus species group (Padial et al. 2014, 2016; de Oliveira et al. 2017, 2020; Taucce et al. 2020), 13 species, including P. symptosus, exhibit dor- solateral folds (Duellman and Lehr 2009; Padial et al. 2016). Among these, P adiastolus mainly differs from P. symptosus by more prominent and longer dorsolater- al folds, finger I slightly shorter than finger IH, and skin on dorsal surfaces shagreen (Duellman and Hedges Zoosyst. Evol. 98 (2) 2022, 213-232 2007); P. avicuporum mainly differs by the presence of an interocular dermal fold, fingers bearing lateral fringes, and finely shagreen dorsal skin (Duellman and Pramuk 1999; Duellman and Lehr 2009); P. buccinator mainly differs by continuous and prominent dorsolateral folds, an X-shaped or V-shaped dorsal fold and presence of an interorbital dermal ridge (Rodriguez 1994); P. condor mainly differs by larger male size (SVL 32.1-39.5 mm) and finger I distinctly longer than finger II (Lynch and Duellman 1980); P. conspicillatus mainly differs by finger I being distinctly longer than finger II, fingers bearing lat- eral keels, and dorsal skin finely shagreen (Duellman and Lehr 2009); P. iiap mainly differs by upper eyelid bearing small granules, skin on dorsum coarsely shagreen, basal toe webbing absent, presence of a tarsal fold, and adver- tisement call (Padial et al. 2016); P. malkini mainly dif- fers by finger I distinctly longer than finger IT and more extensive webbing between toes (Lynch 1980; Duellman and Lehr 2009); P. meridionalis mainly differs by fin- ger I being shorter than finger II, dentigerous processes of vomers small and round, and fingers bearing lateral fringes (Duellman and Lehr 2009); P. peruvianus main- ly differs by very prominent and long dorsolateral folds, finger I distinctly longer than finger II, and fingers bear- ing lateral fringes (Kohler 2000; Padial and De la Riva 2009); P. skydmainos mainly differs by more prominent and continuous dorsolateral folds, finger I being shorter than finger II, and a black mid-dorsal tubercle (Flores and Rodriguez 1997; Padial and De la Riva 2005). The close- ly related P. nebulosus mainly differs by skin on dorsum and flanks shagreen, curved supratympanic fold, relative- ly longer legs (TL/SVL 0.64 versus 0.60), presence of a tarsal fold, presence of basal webbing between fingers II and II, bronze-coloured iris in life, advertisement call (see below), and divergence in the mitochondrial 16S gene (p-distance 4.1%). The sister species P. bipunctatus is most similar to P. symptosus in sharing similar adult male size, finger I about equal in length to finger II, tarsus lacking a tarsal fold, fingers lacking lateral keels or fring- es, a pair of enlarged scapular warts, and a similiar colour pattern. However, P. bipunctatus (Fig. 7) differs by skin on dorsum and flanks finely shagreen (tuberculate), blunt- ly rounded snout in dorsal view (subacuminate), basal webbing between toes IV and V (basal webbing between all toes) (Duellman and Hedges 2005; Duellman and Lehr 2009), and considerable divergence in the mitochondrial 16S gene (p-distance 3.3-4.1%). The advertisement call of P. bipunctatus remains unknown. Individuals of the re- cently described species P. pictus, P. pluvian and P. moa from Amazonian Brazil may sometimes exhibit weakly developed dorsolateral folds, but all differ from the new species by shagreen dorsal skin (versus tuberculate) and advertisement call (de Oliveira et al. 2020). Description of the holotype. An adult male, in good state of preservation (Fig. 8), with subgular vocal sac and vocal slits. Head longer than wide (HL/HW = 1.2); snout subacuminate in dorsal view, bluntly rounded in profile; nostrils oriented posterolaterally; canthus rostralis straight 225 Figure 7. Pristimantis bipunctatus (MUSM 31120; GenBank accession number K Y006089) from Yanachaga-Chemillén Na- tional Park, 2290 m a.s.l., Departamento Pasco, Peru, in life: a. Dorsolateral view; b. Ventral view. Photographs by J. Moravec. in dorsal view, sharp in profile; loreal region straight; lips not flared; upper eyelid without tubercles; cranial crests absent. Supratympanic fold prominent, long, almost straight, not covering upper tympanic membrane or an- nulus; tympanic membrane and annulus distinct; tympan- ic membrane nearly slightly higher than long, its length slightly less than half the eye diameter; three prominent postrictal tubercles, conical. Choanae not concealed by palatal shelf of the maxillary arch when roof of mouth is viewed from below; choanae large, round, separated by distance equal to six times diameter of a choana; dentig- erous process of vomers prominent, elongate, not in con- tact, oblique, situated posteromedial to choanae, bearing vomerine teeth; tongue removed for tissue sample; vo- cal sac subgular, vocal slits placed posterolaterally. Skin on dorsum tuberculate, with a pair of prominent conical scapular warts; dorsal surfaces of hind limbs tuberculate, dorsal surfaces of forearms and flanks tuberculate; skin on throat and chest smooth, that on belly areolate; oc- cipital folds absent; dorsolateral folds distinct, but low; discoidal fold conspicuous. Arm without ulnar tubercles; palmar tubercle bifid, flat, equal in size to elongate thenar tubercle; supernumerary tubercles absent; subarticular tubercles prominent, subconical; fingers long and slen- der; finger tips enlarged, rounded on inner two fingers, on two outer fingers ovate, their width about twice the width of digit proximal to disc; circumferential grooves conspicuous, ungual flap very slightly indented on outer fingers; lateral fringes and keels on fingers absent; basal webbing between fingers absent; relative length of fingers Il > IV > II = I; nuptial pad present on dorsal surface of thumb. Toes long and slender (FootL 54% of SVL); heel and tarsus lacking tubercles; tarsal fold absent; inner metatarsal tubercle ovate, prominent, larger than outer; outer metatarsal tubercle round, flat, about one third the size of outer; tarsal fold absent; supernumerary tubercles absent; subarticular tubercles prominent, subconical; nar- row lateral fringes on toes weakly expressed, traces of zse.pensoft.net 226 Kohler, J. et al .: Rediscovery of Pristimantis nebulosus and description of a new species Figure 8. Preserved male holotype of Pristimantis symptosus sp. nov. (MUSM 40256, FGZC 6207) from the Cordillera de Carpish, 2360 ma.s.l., in a. dorsal, and b. ventral views; c. Palmar surface of left hand; d. Head in lateral profile (mirrored). Note prominent scapular tubercle surrounded by black spot. basal toe webbing present; toe tips enlarged, rounded to slightly ovate, their width about 1.5 times the width of toe proximal to disc; circumferential grooves conspicuous; relative length of toes [V > V > III > II > I; toe V reach- ing distal level of penultimate subarticular tubercle of toe IV. Tibiotarsal articulation reaches slightly beyond tip of snout when hind limb flexed parallel to axis of body; heels overlapping when hind limbs flexed perpendicular to axis of body. Measurements (in mm): SVL 27.6; TL 16.8; HL 11.9; HW 9.9; IOD 3.4; ED 3.9; TD 1.7; E-N 3.1; HandL 7.9; FootL 14.8. In life (Fig. 9), dorsal ground colour brown to tan, with dark brown chevrons and irregular dark brown blotches on dorsum, most of them finely outlined with cream; dark brown bars on dorsal surfaces of arms and legs; dark brown interorbital bar, extending to upper eyelids; a pair of black spots surrounding prominent scapular warts; broad black supratympanic stripe; broad blackish canthal zse.pensoft.net stripe; lips dark brown to black, barred with cream; flanks brown with irregular dark brown markings; belly cream; throat and chest orange-brown with dense black mottling; ventral surfaces of thighs and shanks orange tan; poste- rior surface of thighs orange-brown with irregular cream spotting; plantar and palmar surfaces yellowish-brown, densely covered with dark brown mottling; iris copper, with black reticulation; posterior iris periphery pale blue; bones white. After two years in preservative (Fig. 7), the general colour pattern remains the same as in life. Brown ground colouration turned to greyish-tan, with brown flecks, bars and chevrons; black colour of canthal and su- pratympanic stripes faded to anthracite; chest and ventral surfaces of thighs yellowish-cream; belly cream; throat cream with grey mottling. Natural history. The forest at the type locality con- stitutes upper montane rainforest at the transition zone to cloud forest, growing on steep slopes (Fig. 10), with trees not exceeding 20 m height (Jiménez and Pacheco 2016). Zoosyst. Evol. 98 (2) 2022, 213-232 Figure 9. Male holotype of Pristimantis symptosus sp. nov. (MUSM 40256, FGZC 6207) from the Cordillera de Carpish, 2360 m a.s.l., in life: a. Dorsolateral view of left body side; b. Dorsolateral view of right body side; ¢. Ventral view. Note tuberculate skin on dorsum, flanks and forearm. Males were calling from a low position in shrub vegeta- tion along the road during a foggy night and light rain. The holotype was sitting on a leaf approximately 25 cm above the ground. Pristimantis sp. (lacrimosus group) and P. sp. (aff. rhabdocnemus) were found at nearby sites. Nothing else is known. Vocalisation. Calls were recorded at the type locality of P. symptosus on 4 November 2019 (estimated air tem- perature 16 °C). Calling males were easily disturbed and stopped calling when being approached at distances of less than 10 m. Therefore, recordings are of poor quality, as the recorded individual was calling at considerable dis- tance (estimated > 15 m) and there is considerable back- ground noise in the recording. A call voucher could not be collected, but the male holotype was caught at a spot from where the same type of call was emitted (although the in- dividual could not be directly observed calling), making it very probable that the calls described here belong to P. symptosus. Although the poor recording quality hampers access to some call parameters (i.e. pulse structure and exact rate), the call can be described as follows. The ad- vertisement call consists of a single, short, pulsed note, repeated at regular succession (Fig. 11). Calls (= notes) exhibit a very slight upward frequency modulation and are clearly pulsed, although the poor recording quality alas) Figure 10. View of the forest along the road 18A close to the type locality of Pristimantis symptosus sp. nov. in the Cordille- ra de Carpish, Departamento Huanuco, Peru, at approximately 2450 maz.s.l. Photo taken on 4 November 2019. Frequency (kHz) i=) Relative amplitude 0 250 500 750 1000 Time (ms) Figure 11. Audiospectrogram and corresponding oscillogram of one advertisement call allocated to Pristimantis symptosus sp. nov. from Cordillera de Carpish, 2360 m a.s.1., Peru, at 1000 ms time scale. Recording band-pass filtered at 850-6500 Hz. prevents any reliable counting of pulses; however, it can be estimated from some sections of the calls that pulse rate is greater than 200 pulses/second. Numerical param- eters of 23 analysed calls are as follows: call duration (= note duration) 132—186 ms (151.3 + 14.1 ms); inter-call interval 804-1041 ms (882.3 + 79.5 ms); dominant fre- quency 1442-1507 Hz (1468 + 25 Hz), with a second zse.pensoft.net 228 Kohler, J. et al.: Rediscovery of Pristimantis nebulosus and description of a new species peak in call energy of similar intensity at around 2800- 2930 Hz; prevalent bandwidth 1200-4000 Hz; calls were repeated at a rate of 52—63 call/minute. Distribution. Pristimantis symptosus is only known from its type locality and possibly endemic to the Cordil- lera de Carpish. Etymology. The specific epithet is a Latinised adjec- tive derived from the Greek obvpmtwMon (symptosi) mean- ing ‘coincidence’. It refers to the fact that we only dis- covered the new species by coincidence on an unplanned return to the Cordillera de Carpish after forgetting part of our expedition gear there. Discussion As mentioned in the Introduction, the status of Pristiman- tis nebulosus (Henle, 1992) has long been enigmatic. The main reasons for this were an insufficient original species description (Henle 1992) and a poor state of preservation of the holotype. Without any newly collected topotypic Specimens, we would have been unable to evaluate the status of this taxon by examination of the holotype alone (see Duellman and Lehr 2009). A superficial first ex- amination of the type specimen gave the impression of certain differences in body proportions when compared to newly collected topotypic specimens. Only a very de- tailed specimen-to-specimen comparison of the holotype and topotypes, taking into account certain artifacts and damage of the type specimen, finally resulted in a con- vincing allocation of this nominal taxon to a living pop- ulation of Pristimantis. Although we are completely con- vinced, given the congruence in morphological traits, that our collected topotypes are conspecific with the holotype of P. nebulosus, further evidence could be achieved by ob- taining molecular genetic data from the holotype. We tried to extract and amplify DNA from a small piece of thigh muscle of the holotype using standard protocols, but this approach unfortunately failed. The established, but chal- lenging and costly method of target-enriched sequencing of archival DNA from old specimens (e.g. Scherz et al. 2020; Vences et al. 2021) would currently be an option to obtain sequences for the holotype of P. nebulosus, but without any guarantee of success. However, this method remains available as a promising future option for many old type specimens of species with uncertain status cur- rently placed in the species-rich genus Pristimantis. In recent decades, the Cordillera de Carpish in central Peru has been the place of discovery of several new anu- rans and seems to hold a remarkable diversity of endemic species (see Lehr and Rodriguez 2017 for a summary). Our molecular analysis indicated the presence of a new candidate species in the Pristimantis conspicillatus spe- cies group from the Cordillera de Carpish being sister to P. bipunctatus, but considerably divergent from it. The herein described Pristimantis symptosus differs from the holotype of P. bipunctatus (KU 291638, EF493702) originating from east of Oxapampa (at the border of zse.pensoft.net Yanachaga-Chemillén National Park) by 4.1% diver- gence in the 16S fragment. The divergence between nom- inal P. bipunctatus samples from Yanachaga-Chemillén and Pui Pui Forest further south is pronounced (3.7%), indicating a possible complex of species hidden under the name P. bipunctatus. Remarkably, the genetic dis- tance of P. symptosus to P. bipunctatus samples originat- ing from the geographically more distant Pui Pui Forest in the south 1s lower (3.3%) than the genetic distance to P. bipunctatus samples from the geographically closer Yanachaga-Chemillén National Park (3.9-4.1%; see also Fig. 12). Pristimantis symptosus is further divergent from the herein identified and closely related, but morphologi- cally different P. nebulosus by a genetic distance of 4.1%. $*Huanuce or * See Es x MHuancayo \ Figure 12. Satellite image of central Peru (from GoogleEarth) indicating localities of Pristimantis samples included in our mo- lecular analysis: P. nebulosus (light blue), P. symptosus sp. nov. (yellow) and P. bipunctatus (red). In fact, providing unambiguous evidence for the exis- tence of a new species 1s challenging, when based on a sin- gle specimen. Castroviejo-Fisher et al. (2011) discussed the problems of limited sample size in anuran taxonomy. However, in certain cases, the description of so-called sin- gleton species (sensu Lim et al. 2012) is justified and war- ranted (see Kohler and Padial 2016; Scherz 2020). The study of specimens of Pristimantis over the past decades indicates that certain characters, such as relative finger length and dorsal skin texture, are barely variable within species, even across specimens of different sex and age (e.g. Duellman and Lehr 2009). Here, we provided mul- tiple lines of evidence for the distinctiveness of the new species including genetic differentiation, qualitative dif- ferences in morphological characters and advertisement call. Molecular analyses clearly placed the new species in the P. conspicillatus species group, but with considerable differentiation to other species of the group (p-distances Zoosyst. Evol. 98 (2) 2022, 213-232 3.3-17.8% to all other species). The combination of tu- bercular dorsal skin, areolate skin on venter, presence of dorsolateral folds, first finger about equal in length to sec- ond, basal webbing between all toes, and a copper iris in life distinguishes P. symptosus morphologically from all other recognised species in the P. conspicillatus group. Furthermore, the advertisement call differs from all other calls known for species in the P. conspicillatus group (e.g. Kohler 2000; Padial and De la Riva 2009; Padial et al. 2016). However, advertisement calls have been described only for a fraction of species in this group and molecu- lar data for some species allocated to the group are still lacking. The unknown call of P. bipunctatus and several related species argue for future bioacoustic investigations of this taxonomically challenging group of Pristimantis. Nevertheless, given the combination of all lines of evi- dence as described above, we think that the description of this singleton species is justified and warranted. Of course, aspects such as intra-specific variability, sexual dimorphism and natural history will remain unknown un- til further research finds additional individuals of P. symp- tosus, but the data provided with this species description provide the taxonomic foundation for such future studies. Given the considerable genetic differentiation among nominal P. bipunctatus from different localities men- tioned above, the close relationships of P. symptosus and P. nebulosus to these and the presence of rather similar, almost cryptic species like P. adiastolus (see Duellman and Hedges 2007; Duellman and Lehr 2009), we hypoth- esise that the eastern montane forests of central Peru may still hold a variety of undescribed species in the P. con- spicillatus group (with P. bipunctatus from Pui Pui Forest representing one potentially undescribed candidate spe- cies), all very similar in general appearance. Although an investigation of other groups of Pristi- mantis was beyond the aim of this study, we herein doc- ument considerable genetic differentiation within the P. danae species group. As already revealed by former studies (Moravec et al. 2020), there is marked genetic differentiation in the 16S gene fragment among samples considered to represent P. reichlei (Fig. 4). The inclusion of our samples tentatively identified as P. cf. reichlei from Panguana (ca. 230-260 m a.s.l.), Departamento Huanu- co, showed these samples to be highly divergent from and, perhaps, not even the phylogenetically closest rela- tives of P. reichlei, with p-distances of 8.7% to topotypic P. reichlei from Bolivia (Los Guacharos, Departamento Cochabamba; Padial and De la Riva 2009), and 8.1—-8.5% to other P. reichlei from different Peruvian localities. We, therefore, flag the respective population occurring at Pan- guana as a new candidate species whose status will be investigated in a forthcoming study. Acknowledgements We are indebted to the Servicio Nacional Forestal y de Fauna Silvestre (SERFOR), particularly to Ana Luisa 229 Calderon Valenzuela, Rat Javier Dancé Sifuentes and Victor Vargas Garcia, for dealing with our research proposals and issuing research and collection permits (RGD 071-2020-MINAGRI-SERFOR-DGGSPFFS and D000067-2021-MINAGRI-SERFOR-DGGSPFFS). 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Journal of Herpetology 54(2): 245-257. https://doi.org/10.1670/19-114 Vences M, Kohler J, Andreone F, Craul A-K, Crottini A, du Preez L, Preick M, Rancilhac L, Rédel M-O, Scherz MD, Streicher JW, Hofreiter M, Glaw F (2021) Target-enriched DNA sequencing clarifies the identity of name-bearing types of the Gephyromantis plicifer complex and reveals a new species of mantellid frog from Madagascar (Amphibia, Anura). Spixiana 44: 175-202. https:// pfeil-verlag.de/wp-content/uploads/2021/12/SPX_44-2 11 VE. pdf Zumel D, Buckley D, Ron SR (2021) The Pristimantis tachyblepharis species group, a clade of miniaturized frogs: Description of four new species and insight into the evolution of body size in the genus. Zoological Journal of the Linnean Society 195(1): 315-354. https:// doi.org/10.1093/zoolinnean/zlab044 Table A1. List of Pristimantis samples plus Yunganastes pluvicanorus (outgroup) used for the molecular analysis of the 16S gene fragment, including voucher number, GenBank accession number and locality. Order of samples are as they appear in Figure 4 from top to bottom. Accession numbers in bold refer to newly obtained sequences for this study. Species Voucher specimen GenBank accession number Locality P. bipunctatus NMP6V/75062 KY006090 Peru: Junin: Pui Pui Forest, 1700 m a.s.I. P. bipunctatus MUSM 31179 KY594758 Peru: Junin: Pui Pui Forest, 1615 m a.s.l. P. bipunctatus KU 291638 EF493702 Peru: Pasco: East of Oxapampa, 2120 ma.s.l. P. bipunctatus MUSM 31120 KY006089 Peru: Pasco: PN Yanachaga-Chemillén, 2290 m a.s.l. P. nebulosus MUSM 40299, FGZC 6254 ON710982 Peru: Huanuco: Abra La Divisoria, 1650 m a.s.l. P. nebulosus MUSM 40298, FGZC 6253 ON710981 Peru: Huanuco: Abra La Divisoria, 1650 m a.s.l. P. nebulosus MUSM 40326, FGZC 6281 ON710983 Peru: Huanuco: Abra La Divisoria, 1650 m a.s.l. P. symptosus MUSM 40256, FGZC 6207 ON710980 Peru: Huanuco: Cordillera de Carpish, 2360 m a.s.l. P. cf. skydmainos ZSM 1980/2008 ON710985 Peru: Huadnuco: Panguana P. skydmainos MHNSM 10071 EF493393 Peru P. gutturalis 57 PG JN691313 French Guiana: Mitaraka P. incertus - EU186650 Trinidad and Tobago: Tobago: King’s Bay Reservoir P. zeuctotylus 209 AF JN691259 French Guiana: Angouleme P. vilarsi AJC 3944 KP149278 Colombia: Meta: Finca Los Almendros P. fenestratus CFBHT 05708 KU495458 Brazil: Amazonas: Manaus P. fenestratus INPAH 20931 KY712673 Brazil: Amazonas: Borba P. koehleri MNCN 42990 EU192278 Bolivia: Santa Cruz: Angostura-Samaipata road P. samaipatae MNCN 42987 EU192292 Bolivia: Santa Cruz: Angostura-Samaipata road zse.pensoft.net 232 Species P. latro P. chiastonotus P. vinhai P. palodutrai P. ramagil P. lymani P. achatinus P. malkini P. malkini P. citriogaster P. condor P. conspicillatus P. buccinator P. buccinator P. rupicola P. gaigeae P, pulchridormientes P. pluvialis P. lacrimosus P. bromeliaceus P. lanthanites P. colomai P. toftae P, rhabdolaemus P. danae P. danae P. reichlei P. reichlei P. reichlei P. reichlei P. cf. reichlei P. cf. reichlei P. cf. reichlei P. cf. reichlei P. cf. reichlei Y, pluvicanorus zse.pensoft.net Voucher specimen LZATM 467 162AF UFMG 19856 MNRJ 40299 MNRJ 50249 KU 218019 QCAZ 62884 QCAZ 25544 QCAZ 28296 KU 212278 KU 217857 QCAZ 28448 MNCN-DNA 9504 MUSM 33269 UFMG 20568 KRL 1079 CORBIDI 15566 CORBIDI 11862 QCAZ 40261 QCAZ16699 KU 222001 QCAZ 17101 KU 215493 MNCN 43036 MNCN 44234 MNKA 7273 CORBIDI 16219 MNCN 43249 MNCN 43012 IDLR 4779 ZSM 1985/2008 ZSM 1986/2008 FGZC 5342 FGZC 6334 FGZC 3300 MNCN-DNA 6004 GenBank accession number KX242519 EU201061 MN954211 JX267360 JX267319 EF493392 MK829664 MH516185 EU186663 EF493700 EF493701 EF493529 EU712630 KY652650 MN954206 FJ784461 KX664107 KX155577 MT636524 MT636527 EF493695 EF493354 EF493353 EU192257 EU192270 EU192265 KY652657 EU192288 EU192287 EU192285 ON710986 ON710987 ON710989 ON710984 ON710988 EU192247 Kohler, J. et al.: Rediscovery of Pristimantis nebulosus and description of a new species Locality Brazil: Para: Anapu Suriname: Brownsberg Brazil: Bahia: PN Serra das Lontras Brazil: Bahia: Canavieiras Brazil: Alagoas: Murici Ecuador: Loja: 3.9 km E Loja Ecuador: S. D. de los Tsachilas: Valle Hermoso Ecuador: Napo: Jatun Sacha Ecuador: Sucumbios: Monte Tour Peru: San Martin: 14 km NE Tarapoto Ecuador: Morona Santiago: N of Galaquiza Ecuador: Sucumbios Peru: Cuzco: Blanquillo Peru: Madre de Dios: Los Amigos Biological Station Brazil: Bahia: Serra do Buriti do Ouro Panama: El Copé: PN Omar Torrijos Peru: Huanuco: PN Tingo Maria Peru: Cuzco: Kosnipata near Chontacha Ecuador: Pastaza Ecuador: Morona Santiago Peru: Loreto: San Jacinto Ecuador: Esmeraldas: Alto Tambo Peru: Madre de Dios: 15 km E Puerto Maldonado Bolivia: Santa Cruz: La Yunga de Mairana Peru: Cuzco: Union, Kosnipata valley Bolivia: La Paz: Serrania Bella Vista Peru: Cuzco: Kosnipata valley Peru: Cuzco: 5 km from San Lorenzo to Qince Mil Bolivia: Cochabamba: Los Guacharos Peru: Puno: entre Puerto Leguia y San Gaban Peru: Huanuco: Panguana Peru: Huanuco: Panguana Peru: Huanuco: Panguana Peru: Huanuco: Panguana Peru: Huanuco: Panguana Bolivia: Cochabamba: Penstock bridge